A human stem cell-derived test system for agents modifying neuronal N-methyl-D-aspartate-type glutamate receptor Ca2+-signalling.

Methods to assess neuronal receptor functions are needed in toxicology and for drug development. Human-based test systems that allow studies on glutamate signalling are still scarce. To address this issue, we developed and characterized pluripotent stem cell (PSC)-based neural cultures capable of forming a functional network. Starting from a stably proliferating neuroepithelial stem cell (NESC) population, we generate "mixed cortical cultures" (MCC) within 24 days. Characterization by immunocytochemistry, gene expression profiling and functional tests (multi-electrode arrays) showed that MCC contain various functional neurotransmitter receptors, and in particular, the N-methyl-D-aspartate subtype of ionotropic glutamate receptors (NMDA-R). As this important receptor is found neither on conventional neural cell lines nor on most stem cell-derived neurons, we focused here on the characterization of rapid glutamate-triggered Ca2+ signalling. Changes of the intracellular free calcium ion concentration ([Ca2+]i) were measured by fluorescent imaging as the main endpoint, and a method to evaluate and quantify signals in hundreds of cells at the same time was developed. We observed responses to glutamate in the low µM range. MCC responded to kainate and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and a subpopulation of 50% had functional NMDA-R. The receptor was modulated by Mg2+, Zn2+ and Pb2+ in the expected ways, and various toxicologically relevant agonists (quinolinic acid, ibotenic acid, domoic acid) triggered [Ca2+]i responses in MCC. Antagonists, such as phencyclidine, ketamine and dextromethorphan, were also readily identified. Thus, the MCC developed here may fill an important gap in the panel of test systems available to characterize the effects of chemicals on neurotransmitter receptors.

Recording physiological history of cells with chemical labeling.

Recordings of the physiological history of cells provide insights into biological processes, yet obtaining such recordings is a challenge. To address this, we introduce a method to record transient cellular events for later analysis. We designed proteins that become labeled in the presence of both a specific cellular activity and a fluorescent substrate. The recording period is set by the presence of the substrate, whereas the cellular activity controls the degree of the labeling. The use of distinguishable substrates enabled the recording of successive periods of activity. We recorded protein-protein interactions, G protein-coupled receptor activation, and increases in intracellular calcium. Recordings of elevated calcium levels allowed selections of cells from heterogeneous populations for transcriptomic analysis and tracking of neuronal activities in flies and zebrafish.